Botulinum neurotoxins (“BoNTs”) are a family of structurally similar proteins that cause peripheral neuromuscular blockade and respiratory paralysis. BoNTs are exceedingly toxic, possessing an extremely low LD50 (1-50 ng/kg) (National Institute of Occupational Safety and Health, Registry of Toxic Effects of Chemical Substances (R-TECS), Cincinnati, Ohio: National Institute of Occupational Safety and Health, 1996). There are seven major BoNT serotypes (BoNT A-G) and multiple subtypes (Smith et al., “Sequence Variation Within Botulinum Neurotoxin Serotypes Impacts Antibody Binding and Neutralization,” Infect. Immun. 73(9):5450-5457 (2005)), but all have common structural features and a similar mechanism of action. BoNTs are synthesized as single chain propeptides with intramolecular disulfide bonds (Mr approximately 150,000; approximately 1,300 amino acids) with extensive areas of sequence homology. The majority are activated by proteolytic cleavage to generate a disulfide-bonded heterodimer containing light (approximately 50 kDa) and heavy (approximately 100 kDa) chains (“LC” and “HC” respectively).
Botulinum neurotoxin serotype A (“BoNT/A”) heterodimer has been extensively studied and has been found to contain three functional domains. Toxicity is associated with metalloprotease activity confined to the LC, neuron binding activity is associated with the C-terminal half of the HC (HCC), and translocation activity responsible for delivering the LC protease to the neuronal cytosol is associated with the N-terminal half of the HC (HCN) (Johnson, “Clostridial Toxins as Therapeutic Agents: Benefits of Nature's Most Toxic Proteins,” Annu. Rev. Microbiol. 53:551-575 (1999); Montecucco et al., “Structure and Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys. 28(4):423-472 (1995)).
The toxicity of BoNTs is a consequence of a multi-step mechanism culminating in a LC-mediated proteolytic event that disrupts the neuronal machinery for synaptic vesicle exocytosis. During BoNT poisoning, BoNTs must first cross epithelial barriers by transcytosis (Simpson, “Identification of the Major Steps in Botulinum Toxin Action,” Annu. Rev. Pharmacol. Toxicol. 44:167-193 (2004)). The BoNT then passes into the circulation by an unknown pathway, from which it selectively targets the presynaptic membrane of motor neurons at neuromuscular junctions. Toxicity at the neuromuscular junction involves (i) binding to the plasma membrane, (ii) internalization into endocytic vesicles, (iii) activation within an acidic endosomal compartment that enables HC-mediated translocation of the LC into the neuronal cytoplasm, and (iv) catalytic cleavage by the LC zinc-endopeptidase of protein components in the neuronal machinery required for synaptic vesicle exocytosis.
The endopeptidase activity responsible for toxicity is associated with a HExxHxxH (SEQ ID NO:1) motif in the LC which is characteristic of the thermolysin family of metalloproteases. Mutagenesis experiments with the BoNT/A light chain have identified the minimal essential domain for toxicity (Kurazono et al., “Minimal Essential Domains Specifying Toxicity of the Light Chains of Tetanus Toxin and Botulinum Neurotoxin Type A,” J. Biol. Chem. 267(21):14721-14729 (1992)), and have pinpointed the amino acids involved in Zn2+ coordination at the metalloprotease active site (Rigoni et al., “Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A,” Biochem. Biophys. Res. Commun. 288(5):1231-1237 (2001)). These data are corroborated by crystallography-based structures currently available for the majority of BoNT serotypes, and by crystallographic data for LC/LC mutants expressed as single entities, or co-crystallized with the substrate or inhibitors (Lacy et al., “Crystal Structure of Botulinum Neurotoxin Type A and Implications for Toxicity,” Nat. Struct. Biol. 5(10):898-902 (1998); Breidenbach et al., “Substrate Recognition Strategy for Botulinum Neurotoxin Serotype A,” Nature 432:925-929 (2004); Fu et al., “Light Chain of Botulinum Neurotoxin Serotype A: Structural Resolution of a Catalytic Intermediate,” Biochemistry 45:8903-8911 (2006); Garcia-Rodriguez et al., “Molecular Evolution of Antibody Cross-Reactivity for Two Subtypes of Type A Botulinum Neurotoxin,” Nat. Biotechnol. 25:107-116 (2007); Burnett et al., “Inhibition of Metalloprotease Botulinum Serotype A From a Pseudo-Peptide Binding Mode to a Small Molecule That is Active in Primary Neurons,” J. Biol. Chem. 282:5004-5014 (2007); Silvaggi et al., “Structures of Clostridium botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility,” Chem. Biol. 14(5):533-542 (2007); Silvaggi et al., “Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex,” Biochemistry 47(21):5736-5745 (2008); Zuniga et al., “A Potent Peptidomimetic Inhibitor of Botulinum Neurotoxin Serotype A Has a Very Different Conformation than SNAP-25 Substrate,” Structure 16:588-1597 (2008); Kumaran et al., “Structure- and Substrate-Based Inhibitor Design for Clostridium botulinum Neurotoxin Serotype A,” J. Biol. Chem. 283:18883-18891 (2008); Kumaran et al., “Substrate Binding Mode and Its Implication on Drug Design for Botulinum Neurotoxin A,” PloS Pathog. 4(9):e1000165 (2008); Swaminathan et al., “Structural Analysis of the Catalytic and Binding Sites of Clostridium botulinum Neurotoxin B,” Nat. Struct. Biol. 7(8):693-699 (2000); Hanson et al., “Cocrystal Structure of Synaptobrevin-II Bound to Botulinum Neurotoxin Type B at 2.0 Å Resolution,” Nat. Struct. Biol. 7(8):687-692 (2000); Eswaramoorthy et al., “Novel Mechanism for Clostridium botulinum Neurotoxin Inhibition,” Biochemistry 41:9795-9802 (2002); Eswaramoorthy et al., “Role of Metals In the Biological Activity of Clostridium botulinum Neurotoxins,” Biochemistry 43(8):2209-2216 (2004); Jin et al., “Structural and Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity,” Biochemistry 46:10685-10693 (2007); Arndt et al., “Structure of Botulinum Neurotoxin Type D Light Chain at 1.65 Å Resolution: Repercussions for VAMP-2 Substrate Specificity,” Biochemistry 45:3255-3262 (2006); Agarwal et al., “Structural Analysis of Botulinum Neurotoxin Type E Catalytic Domain and Its Mutant Glu212>Gln Reveals the Pivotal Role of the Glu212 Carboxylate in the Catalytic Pathway,” Biochemistry 43:6637-6644 (2004); Agarwal et al., “Analysis of Active Site Residues of Botulinum Neurotoxin E By Mutational, Functional, and Structural Studies: Glu335>Gln is an Apoenzyme,” Biochemistry 44:8291-8302 (2005); Agarwal et al., “SNAP-25 Substrate Peptide (Residues 180-183) Binds to But Bypasses Cleavage by Catalytically Active Clostridium botulinum Neurotoxin E,” J. Biol. Chem. 283:25944-25951 (2008); Agarwal et al., “Structural Analysis of Botulinum Neurotoxin Serotype F Light Chain: Implications on Substrate Binding and Inhibitor Design,” Biochemistry 44:11758-11765 (2005); Agarwal et al., “Mode of VAMP Substrate Recognition and Inhibition of Clostridium botulinum Neurotoxin F,” Nat. Struct. Mol. Biol. 16:789-794 (2009); Arndt et al., “Crystal Structure of Botulinum Neurotoxin Type G Light Chain: Serotype Divergence In Substrate Recognition,” Biochemistry 44:9574-9580 (2005)).
Recombinant BoNT proteins or peptides have been reported for several serotypes, primarily as part of efforts aimed at developing a vaccine against BoNT poisoning. The receptor-binding HC domain (HCC) has been produced in a variety of expression systems. These recombinant HC preparations were effective immunogens and protected animals challenged with wt BoNTs (Byrne et al., “Development of Vaccines for Prevention of Botulism,” Biochimie 82:955-966 (2000); Ravichandran et al., “Trivalent Vaccine Against Botulinum Toxin Serotypes A, B, and E That Can Be Administered By the Mucosal Route,” Infect. Immun. 75(6):3043-3054 (2007); Baldwin et al., “Subunit Vaccine Against the Seven Serotypes of Botulism,” Infect. Immun. 76(3):1314-131 (2008); Smith, “Development of Recombinant Vaccines for Botulinum Neurotoxin,” Toxicon 36:1539-1548 (1998); Baldwin et al., “Characterization of the Antibody Response to the Receptor Binding Domain of Botulinum Neurotoxin Serotypes A and E,” Infect Immun. 73(10):6998-7005 (2005); Woodward et al., “Expression of HC Subunits from Clostridium botulinum Types C and D and Their Evaluation as Candidate Vaccine Antigens In Mice,” Infect. Immun. 71(5):2941-2944 (2003); Webb et al., “Protection with Recombinant Clostridium botulinum C1 and D Binding Domain Subunit (Hc) Vaccines Against C and D Neurotoxins,” Vaccine 25(21):4273-4282 (2007); Lee et al., “C-Terminal Half Fragment (50 kDa) of Heavy Chain Components of Clostridium botulinum Type C and D Neurotoxins Can Be Used As an Effective Vaccine,” Microbiol. Immunol. 51(4):445-455 (2007); LaPenotiere et al., “Expression of a Large, Nontoxic Fragment of Botulinum Neurotoxin Serotype A and Its Use As an Immunogen,” Toxicon 33(10):1383-1386 (1995); Clayton et al., “Protective Vaccination with a Recombinant Fragment of Clostridium botulinum Neurotoxin Serotype A Expressed From A Synthetic Gene In Escherichia coli,” Infect. Immun. 63(7):2738-2742 (1995); Byrne et al., “Purification, Potency, and Efficacy of the Botulinum Neurotoxin Type A Binding Domain from Pichia pastoris As a Recombinant Vaccine Candidate,” Infect Immun. 66(10):4817-4822 (1998); Lee et al., “Candidate Vaccine Against Botulinum Neurotoxin Serotype A Derived From a Venezuelan Equine Encephalitis Virus Vector System,” Infect. Immun. 69(9):5709-5715 (2001); Maddaloni et al., “Mucosal Vaccine Targeting Improves Onset of Mucosal and Systemic Immunity to Botulinum Neurotoxin A,” J. Immunol. 177(8):5524-5532 (2006); Yu et al., “The Recombinant He Subunit of Clostridium botulinum Neurotoxin Serotype A Is an Effective Botulism Vaccine Candidate,” Vaccine 27(21):2816-2822 (2009); Boles et al., “Recombinant C Fragment of Botulinum Neurotoxin B Serotype (rBoNTB (HC)) Immune Response and Protection In the Rhesus Monkey,” Toxicon 47(8):877-884 (2006); Zeng et al., “Protective Immunity Against Botulism Provided By a Single Dose Vaccination With an Adenovirus-Vectored Vaccine,” Vaccine 25(43):7540-7548 (2007); Xu et al., “An Adenoviral Vector-Based Mucosal Vaccine Is Effective In Protection Against Botulism,” Gene Ther. 16(3):367-375 (2009); Byrne et al., “Fermentation, Purification, and Efficacy of a Recombinant Vaccine Candidate Against Botulinum Neurotoxin Type F From Pichia pastoris,” Protein Expr. Purif. 18(3):327-337 (2000); Holley et al., “Cloning, Expression and Evaluation of a Recombinant Sub-Unit Vaccine Against Clostridium botulinum Type F Toxin,” Vaccine 19(2-3):288-297 (2000); Foynes et al., “Vaccination Against Type F Botulinum Toxin using Attenuated Salmonella enterica var typhimurium Strains Expressing the BoNT/F H(C) Fragment,” Vaccine 21(11-12):1052-1059 (2003); Yu et al., “Evaluation of a Recombinant He of Clostridium botulinum Neurotoxin Serotype F As an Effective Subunit Vaccine,” Clin. Vaccine Immunol. 15(12):1819-1823 (2008)). Recombinant HCC was additionally demonstrated to retain the ability to transcytose epithelia, thereby providing effective immunogen delivery by inhalation (Baldwin et al., “Subunit Vaccine Against the Seven Serotypes of Botulism,” Infect. Immun. 76(3):1314-131 (2008)). Enzymatically active and inactive recombinant LC derivatives have also been expressed (Kurazono et al., “Minimal Essential Domains Specifying Toxicity of the Light Chains of Tetanus Toxin and Botulinum Neurotoxin Type A,” J. Biol. Chem. 267(21):14721-14729 (1992); Rigoni et al., “Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A,” Biochem. Biophys. Res. Commun. 288(5):1231-1237 (2001); Breidenbach et al., “Substrate Recognition Strategy for Botulinum Neurotoxin Serotype A,” Nature 432:925-929 (2004); Fu et al., “Light Chain of Botulinum Neurotoxin Serotype A: Structural Resolution of a Catalytic Intermediate,” Biochemistry 45:8903-8911 (2006); Silvaggi et al., “Structures of Clostridium botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility,” Chem. Biol. 14(5):533-542 (2007); Kumaran et al., “Structure- and Substrate-Based Inhibitor Design for Clostridium botulinum Neurotoxin Serotype A,” J. Biol. Chem. 283:18883-18891 (2008); Zhou et al., “Expression and Purification of the Light Chain of Botulinum Neurotoxin A: A Single Mutation Abolishes Its Cleavage of SNAP-25 and Neurotoxicity After Reconstitution With the Heavy Chain,” Biochemistry 34(46):15175-15181 (1995); Li et al., “High-Level Expression, Purification, and Characterization of Recombinant Type A Botulinum Neurotoxin Light Chain,” Protein Expr. Purif. 17(3):339-344 (1999); Kadkhodayan et al., “Cloning, Expression, and One-Step Purification of the Minimal Essential Domain of the Light Chain of Botulinum Neurotoxin Type A,” Protein Expr. Purif. 19(1):125-130 (2000); Ahmed et al., “Light Chain of Botulinum A Neurotoxin Expressed As an Inclusion Body From a Synthetic Gene Is Catalytically and Functionally Active,” J. Protein Chem. 19(6):475-487 (2000); Li et al., “Probing the Mechanistic Role of Glutamate Residue In the Zinc-Binding Motif of Type A Botulinum Neurotoxin Light Chain,” Biochemistry 39(9):2399-2405 (2000); Ahmed et al., “Enzymatic Autocatalysis of Botulinum A Neurotoxin Light Chain,” J. Protein Chem. 20(3):221-231 (2001); Ahmed et al., “Factors Affecting Autocatalysis of Botulinum A Neurotoxin Light Chain,” Protein J. 23(7):445-451 (2004); Segelke et al., “Crystal Structure of Clostridium botulinum Neurotoxin Protease In a Product-Bound State: Evidence for Noncanonical Zinc Protease Activity,” Proc. Natl. Acad. Sci. (USA) 101(18):6888-6893 (2004); Baldwin et al., “The C-Terminus of Botulinum Neurotoxin Type A Light Chain Contributes to Solubility, Catalysis, and Stability,” Protein Expr. Purif. 37(1):187-195 (2004); Ahmed et al., “Identification of Residues Surrounding the Active Site of Type A Botulinum Neurotoxin Important for Substrate Recognition and Catalytic Activity,” Protein J. 27(3):151-162 (2008)). These have been found to be non-toxic in vivo even when LC enzymatic activity was preserved, because the presence of disulfide-bonded HC is required for BoNT targeting. The LC expressed as a separate entity, or as part of a holotoxoid, is less immunogenic than HC (Smith et al., “Sequence Variation Within Botulinum Neurotoxin Serotypes Impacts Antibody Binding and Neutralization,” Infect. Immun. 73(9):5450-5457 (2005)).
To achieve LC conformations that more closely resemble the native toxin, and to generate a greater variety of antigens for vaccine design, the enzymatically active endopeptidase constructs representing LC fused to the full and C-terminally truncated version of HCN were also expressed in E. coli (Chaddock et al., “Expression and Purification of Catalytically Active, Non-Toxic Endopeptidase Derivatives of Clostridium botulinum Toxin Type A,” Protein Expr. Purif. 25(2):219-228 (2002); Jensen et al., “Expression, Purification, and Efficacy of the Type A Botulinum Neurotoxin Catalytic Domain Fused to Two Translocation Domain Variants,” Toxicon 41(6):691-701 (2003); Sutton et al., “Preparation of Specifically Activatable Endopeptidase Derivatives of Clostridium botulinum Toxins Type A, B, and C and Their Applications,” Protein Expr. Purif. 40(1):31-41 (2005)). With subsequent improvements in the constructs and the expression system, these derivatives were used as building blocks to re-target specificity of botulinum neurotoxin A through substitution of native HCC with wheat germ agglutinin (WGA), NGF, and EGF (Chaddock et al., “Inhibition of Vesicular Secretion In Both Neuronal and Nonneuronal Cells By a Retargeted Endopeptidase Derivative of Clostridium botulinum Neurotoxin Type A,” Infect. Immun. 68(5):2587-2593 (2000); Chaddock et al., “A Conjugate Composed of Nerve Growth Factor Coupled to a Non-Toxic Derivative of Clostridium botulinum Neurotoxin Type A Can Inhibit Neurotransmitter Release In vitro,” Growth Factors 18(2):147-155 (2000); Duggan et al., “Inhibition of Release of Neurotransmitters From Rat Dorsal Root Ganglia By a Novel Conjugate of a Clostridium botulinum Toxin A Endopeptidase Fragment and Erythrina cristagalli Lectin,” J. Biol. Chem. 277(38):34846-34852 (2002); Chaddock et al., “Retargeted Clostridial Endopeptidases: Inhibition of Nociceptive Neurotransmitter Release In vitro, and Antinociceptive Activity In In vivo Models of Pain,” Mov. Disord. Suppl 8:S42-S47 (2004); Foster et al., “Re-Engineering the Target Specificity of Clostridial Neurotoxins—A Route to Novel Therapeutics,” Neurotox. Res. 9(2-3):101-107 (2006)).
Several laboratories have reported expressing recombinant, full-length BoNTs in E. coli. Rummel et al., “Two Carbohydrate Binding Sites in the HCC-Domain of Tetanus Neurotoxin Are Required for Toxicity,” J. Mol. Biol. 326(3):835-847 (2003); Rummel et al., “The HCC-Domain of Botulinum Neurotoxins A and B Exhibits a Singular Ganglioside Binding Site Displaying Serotype Specific Carbohydrate Interaction,” Mol. Microbiol. 51(3):631-643 (2004); Rummel et al., “Synaptotagmins I and II Act as Nerve Cell Receptors for Botulinum Neurotoxin G,” J. Biol. Chem. 279(29):30865-30870 (2004); and Bade et al., “Botulinum Neurotoxin Type D Enables Cytosolic Delivery of Enzymatically Active Cargo Proteins to Neurons Via Unfolded Translocation Intermediates,” J. Neurochem. 91(6):1461-1472 (2004), described the expression of full-length single chain BoNT/G, /D, /B, and /A in E. coli, either as the wt, or with a thrombin-specific cleavage site inserted between the HC and LC, or with the LC protease inactivated by a point mutation. Kiyatkin et al., “Induction of an Immune Response by Oral Administration of Recombinant Botulinum Toxin,” Infect. Immun. 65:4586-4591 (1997), reported the expression of BoNT/C in E. coli, with three inactivating point mutations (H229>G; E230>T; H233>N) in the LC protease, without the insertion of any specific proteolytic cleavage site between the LC and HC. There was no evidence that this BoNT/C single chain was processed into a disulfide-bonded heterodimer in vivo, but it was effective as an immunogen when orally administered. In all reports, the single chain holotoxin expressed in E. coli was not secreted into the culture medium or periplasm and had to be recovered from whole cell lysates. Expression problems in E. coli are associated with improper protein folding stemming from the reducing environment in the E. coli cytosol, the tendency of E. coli to segregate unfolded recombinant proteins within aggregates of inclusion bodies, proteolytic degradation, and a strong codon bias against AT-rich clostridial genes.
Recently, a recombinant, atoxic BoNT/A holotoxoid was expressed in the non-toxic strain of Clostridium botulinum, LNT01, with a yield of approximately 1 mg/L (Pier et al., “Recombinant Holotoxoid Vaccine Against Botulism,” Infect. Immun. 76(1):437-442 (2008)). This recombinant holotoxoid had the mutations R364>A and Y366>F introduced into the LC (BoNT/ARYM), and lacked the ability to cleave the substrate SNAP 25 in vitro. Mice were challenged with up to 1 μg of this derivative (approximately 3.3×104 mouse LD50) and monitored for 96 hours. All mice survived challenge with 1 μg of single-chain or trypsin-nicked dichain of BoNT/ARYM. Immunization with this holotoxoid effectively protected mice against lethal BoNT/A challenge. Although this report is encouraging, no information has yet been provided regarding the physiological trafficking of BoNT/ARYM in comparison with wt BoNT/A.
The most recent report describes the production of catalytically inactive BoNT/A holoprotein (H223>A; E224>A; H227>A) in P. pastoris (ciBoNT/A HP) (Webb et al., “Production of Catalytically Inactive BoNT/A1 Holoprotein and Comparison With BoNT/A1 Subunit Vaccines Against Toxin Subtypes A1, A2, and A3,” Vaccine 27(33):4490-4497 (2009)). The protein expressed from the synthetic gene, which was optimized for codon bias in the host, accumulated intracellularly. There was no introduction of an artificial cleavage site into the loop between LC and HC in the propeptide. The protein was purified in several steps with conventional ion exchange chromatographies. The yield of highly purified product was reported to be approximately 1 milligram from four grams of the frozen methylotrophic yeast. ciBoNT/A HP provided excellent protective immunity, not only against the homologous toxin, but also against two distinct toxin subtypes with significant amino acid divergence. Mice challenged with 50 μg of this derivative (approximately 1.7×106 mouse LD50) and monitored for 240 hours did not display discernible signs of BoNT intoxication.
The selectivity of BoNT targeting to neurons has led several laboratories to consider using BoNT-based molecular vehicles for delivering therapeutic agents. Early work reported that the HC and LC of wt BoNTs could be separated, and that the wt HC could be reconstituted in vitro with either wt LC, or with recombinant LC which could carry point mutations, such as His227>Tyr, which rendered the LC atoxic (Zhou et al., “Expression and Purification of the Light Chain of Botulinum Neurotoxin A: A Single Mutation Abolishes Its Cleavage of SNAP-25 and Neurotoxicity After Reconstitution With the Heavy Chain,” Biochemistry 34(46):15175-15181 (1995); Maisey et al., “Involvement of the Constituent Chains of Botulinum Neurotoxins A and B In the Blockade of Neurotransmitter Release,” Eur. J. Biochem. 177(3):683-691 (1988); Sathyamoorthy et al., “Separation, Purification, Partial Characterization and Comparison of the Heavy and Light Chains of Botulinum Neurotoxin Types A, B, and E,” J. Biol. Chem. 260(19):10461-10466 (1985)). The reconstituted BoNT holotoxin derivatives had a severely reduced ability to transport LC into the neuronal cytosol, probably resulting from the harsh conditions required for HC-LC separation and the difficulty of renaturing the protein and reconstituting native disulfide bonds. Attempts have also been made to use isolated wt HC for targeted delivery, by chemically coupling dextran to the HC to provide sites for attaching fluorescent markers or therapeutic agents (Goodnough et al., “Development of a Delivery Vehicle for Intracellular Transport of Botulinum Neurotoxin Antagonists,” FEBS Lett. 513:163-168 (2002)). Although this “semi-synthetic” BoNT derivative was internalized by neurons, the dextran remained localized to the endosomal compartment and the specificity of the uptake was uncertain. Direct chemical or biochemical attachment of cargo molecules to the HC of BoNTs may not be sufficient for achieving cytosolic delivery, because structural features associated with the toxin LC are required for translocation to the cytosol (Baldwin et al., “The C-Terminus of Botulinum Neurotoxin Type A Light Chain Contributes to Solubility, Catalysis, and Stability,” Protein Expr. Purif. 37(1):187-195 (2004); Brunger et al., “Botulinum Neurotoxin Heavy Chain Belt as an Intramolecular Chaperone for the Light Chain,” PLoS Pathog. 3(9):e113 (2007)). Moreover, when chemical methods are used to attach cargo to BoNT toxoids, cargo attachment is not sufficiently selective and, consequently, produces a heterogeneous population of derivatives. These problems limit the utility of chemically labeled BoNTs as probes for definitive demonstration of BoNT trafficking pathways.
Bade et al., “Botulinum Neurotoxin Type D Enables Cytosolic Delivery of Enzymatically Active Cargo Proteins to Neurons Via Unfolded Translocation Intermediates,” J. Neurochem. 91(6):1461-1472 (2004), described recombinant full-length derivatives of BoNT/D as effective delivery vehicles which were expressed in E. coli with or without an inactivating mutation (E230>A) to the LC protease. To evaluate the delivery of prototypic cargo proteins in neuronal cultures, green fluorescent protein (“GFP”), dihydrofolate reductase, firefly luciferase, or BoNT/A LC were fused to the amino terminus of the recombinant BoNT/D holotoxin. Delivery to the cytosol was evaluated by measuring cleavage of the BoNT/D cytoplasmic substrate, synaptobrevin. Dihydrofolate reductase and BoNT/A LC were reported to be effectively delivered. When luciferase or GFP were the cargo, delivery of the corresponding BoNT/D LC catalytic activity to the cytosol was significantly reduced, presumably due to the large size of the cargo (luciferase) or its rigidity (GFP) (Brejc et al., “Structural Basis for Dual Excitation and Photoisomerization of the Aequorea victoria Green Fluorescent Protein,” Proc. Natl. Acad. Sci. (USA) 94(6):2306-1231 (1997); Palm et al., “The Structural Basis for Spectral Variations in Green Fluorescent Protein,” Nat. Struct. Biol. 4(5):361-365 (1997)).
It has proven particularly difficult to successfully engineer translocation of recombinant toxin LCs from an endosomal compartment to the cytosol. This translocation requires acidification of the lumenal milieu, either to trigger a conformational change in the BoNT heterodimer or to enable its interaction with a translocation mediator (Brunger et al., “Botulinum Neurotoxin Heavy Chain Belt as an Intramolecular Chaperone for the Light Chain,” PLoS Pathog. 3(9):e113 (2007); Kamata et al., “Involvement of Phospholipids In the Intoxication Mechanism of Botulinum Neurotoxin,” Biochim. Biophys. Acta. 1199(1):65-68 (1994); Tortorella et al., “Immunochemical Analysis of the Structure of Diphtheria Toxin Shows all Three Domains Undergo Structural Changes at Low pH,” J. Biol. Chem. 270(46):27439-27445 (1995); Tortorella et al., “Immunochemical Analysis Shows All Three Domains of Diphtheria Toxin Penetrate Across Model Membranes,” J. Biol. Chem. 270(46):27446-27452 (1995)). A requirement for cooperation between the BoNT LC and the translocation domain of the HC is supported by evidence demonstrating that a decapeptide motif, common to the HCN of several BoNT serotypes as well as to diphtheria and anthrax toxins, is required for successful translocation of the LC to the cytosol (Ratts et al., “A Conserved Motif in Transmembrane Helix 1 of Diphtheria Toxin Mediates Catalytic Domain Delivery to the Cytosol,” Proc. Natl. Acad. Sci. (USA) 102(43):15635-15640 (2005)). Future development of BoNTs as carrier vehicles will require a deeper understanding of how the LC itself, and its interaction with HCN, contributes to this mechanism.
Although efforts to express recombinant BoNTs have succeeded in producing effective immunogens, which in some cases are competent for epithelial transcytosis, these efforts have not produced recombinant proteins with the structural features required for targeting the neuronal cytosol with the efficiency of wt toxins. These limitations emphasize the importance of selecting an expression system capable of producing full-length BoNT derivatives that retain native toxin structure, disulfide bonding, and physiological trafficking. Also, work from multiple laboratories has clarified how the structural domains of wt botulinum neurotoxin A (BoNT/A) disable neuronal exocytosis, but important questions remain unanswered. Because BoNT/A intoxication disables its own uptake, wt light chain does not accumulate in neurons at detectable levels.
The present invention is directed to overcoming these and other limitations in the art.